Basement membrane is a thin, dense, sheet-like extracellular matrix that encircles most tissues and provides structural support for epithelial and endothelial cells. Understanding how specialized invasive cells cross basement membrane is of profound importance to human health: Basement membrane transmigration underlies the dispersal of cells in many developmental processes, is required in leukocytes for immune surveillance and inappropriate manifestation of cell-invasive behavior underpins the development of metastatic cancers. F-actin-rich, cell membrane associated structures, termed invadosomes, were identified over three decades ago in normally invasive cell types and metastatic cancer cell lines. Invadosomes are thought to mediate the ability of cells to invade through basement membrane barriers. Owing to the difficulty of examining cell-invasive behavior in vivo, invadosomes have only been studied in vitro, where cell culture conditions do not recapitulate native environmental signals or matrix conditions. As a result, the relevance, regulation and potential functions of invadosomes in vivo remain one of the most critical gaps in our understanding of cell- invasive behavior. Anchor cell invasion in C. elegans is a simple, highly stereotyped in vivo model of cell invasion through basement membrane that uniquely combines single-cell visual and genetic analysis. Through the development of live-cell imaging approaches, our group has recently identified dynamic invadosome structures within the anchor cell that breach the basement membrane. We find that when one invadosome penetrates the BM, new invadosome formation ceases, and a single invasive protrusion matures from the infiltrated invadosome. Using optical highlighting of basement membrane components, we have found that this protrusion pushes the basement membrane aside to clear a path for invasion. The goal of this application is to uncover the mechanistic details of how these invadosomes are used to breach basement membrane. Our proposed research will combine live-cell imaging with genetic and molecular analysis to determine: (1) how key pathways that regulate anchor cell invasion coordinately regulate the formation, dynamics and function of invadosomes prior to and during BM penetration, (2) the role of netrin signaling in selecting a single invadosome for basement membrane invasion, (3) the function of the actin regulator Ena/VASP in promoting basement membrane gap expansion at the anchor cell-basement membrane contact points after invadosome penetration. Together, this work will identify new mechanisms underlying cell invasion and elucidate how they function together to breach basement membrane. This project is relevant to NIH's mission because it will lead to specific therapeutic strategies to control invasive behavior in diseases such as metastatic cancer. PUBLIC HEALTH RELEVANCE: Cell invasion through basement membrane barriers occurs during the dispersal of cells in many normal physiological processes. In human diseases such as metastastic cancer, cell invasion is one of the most clinically relevant yet least understood aspects of cancer progression. The proposed research will investigate the mechanisms that control the subcellular structures that allow invasive cells to penetrate through basement membrane barriers. This work will reveal new molecular targets and strategies to develop more effective and specific treatments to control cell invasive behavior in important human diseases such as cancer.